Our specific aims for the next year will be to develop technology utilizing hybrid plasmid DNAs for the preparation of large quantities of homogeneous glycolytic mRNAs and to determine the precise location and orientation of the structural genes within the hybrid plasmid DNAs. It is our further aim to determine if these isolated glycolytic genes share common DNA sequences and finally to transcribe the hybrid plasmid DNAs in vitro with purified yeast polymerases I, II and III, with particular emphasis on determining the location of initiation and termination sites within the template. The latter studies will be carried out with hybrid plasmids containing glycolytic genes, as well as with hybrid plasmids (recently isolated in our laboratory) containing the genes for yeast 5S, 17S, and 25S ribosomal RNA.